Immune stimulating compound

ABSTRACT

The present invention provides immune stimulating macrolide of formula (I). The macrolide has utility in treating intracellular bacterial, fungal, and protozoal infections.

FIELD OF THE INVENTION

The present invention provides a novel macrolide compound capable ofstimulating the immune system. The present invention relates to a novelcompound for use in medicine, notably in the treatment of intracellularbacterial, fungal, and protozoal infections and in the co-treatment ofviral disease, chronic inflammatory conditions, and cancer whenstimulation of the immune system is beneficial. The compound may also beused as immune modulating adjuvants in vaccination. The novel macrolidemaximizes the modulating effects of the immune system while minimizingthe therapeutically unwanted direct antibacterial effects. The presentinvention also provides methods for preparing the compound of theinvention and for use of the compound in medicine.

BACKGROUND OF THE INVENTION

Intracellular bacterial, fungal, and protozoal infections are often notdiagnosed in healthy individuals as they appear asymptomatic, or becausethe symptoms are mild enough that the infected individual is notinclined to seek medical assistance. As such, intracellular infectionsmay persist latently or may progress to a disease state. Conditionsinterfering with normal T cell function usually leads to progression ofthe disease from a latent infection, and intracellular infections suchas Mycobacterium tuberculosis (Mtb) are a common cause of death inpatients where HIV infection has progressed to AIDS. There is thus agreat need in the art for methods and means of treating intracellularinfections.

Intracellular pathogens such as Mtb have the capacity to hide withinintracellular compartments in monocytes and macrophages causingpersistent infections. Although Mtb are recognized by CD4⁺ T helpercells in the lung and an appropriate response is mounted, the systemfails to create sterilizing immunity (MacMicking 2012).To escape immunerecognition by the host, Mtb have developed a series of mechanism thatinhibits recognition of Mtb peptides presented in the MHC class IIpocket for CD4⁺ T helper cells. Toll like receptor 2 has beendemonstrated to be inhibited by Mtb, which in turn inhibits IFN-γinduced MHC class II expression (Noss 2001). In addition, data suggestthat Mtb has the capacity to inhibit phagosome processing andmaturation, possibly by an invariant chain associated mechanism(Ramachandra 2001). Therefore, the normal antigen processing, loadingand presentation of MHC class II peptides derived from Mtb is impaireddue to Mtb produced immune escape factors.

The endosomal lysosomal pathway is designed to take up pathogens,process them into 12-15 aa long peptides, peptides, that after theremoval of the Invariant chain peptide CLIP by HLA-DM, are loaded intothe MHC class II pocket. The antigen loading is followed by transport ofthe MHC class II-peptide complex to the cell surface for presentationfor the specific T cell receptor of CD4⁺ T helper cells (Roche 2015).Recently the Mtb expressed protein EsxH has been reported to directlyinhibit the endosomal sorting complex required for transport (ESCRT)machinery (Portal-Celhay 2016). EsxH inhibits the ability of antigenpresenting monocytes and macrophages to activate CD4⁺ T helper cells.Since intact ESCRT machinery seems necessary for antigen processing,presentation and activation of T cells, EsxH is the link that explainsMtb induced immune escape by intervening with the MHC class II pathway.

The importance of MHC class II presentation has also been demonstratedin patients with primary immunodeficiencies (PID). PID patients withdefects in the IFN-γ circuit, involving IFNGR and IL-12, have anincreased risk of acquiring TBC and atypical mycobacterial infections.Since MHC class II expression is dependent upon and regulated by IFN-γexpression defects in the IFN-γ circuit will result in additionallydecreased MHC class II expression and a poor activation of CD4⁺ T helpercells.

Protozoa such as Toxoplasma gondii have developed a mechanism to avoidimmune recognition by hiding intracellularly as an obligateintracellular parasite. The mechanism involves interference with MHCclass II expression and thus diminishes the amount of Toxoplasma gondiito be presented for specific CD4⁺ T helper cells. The detailed mechanismis dependent on soluble proteins expressed by Toxoplasma gondii thatinhibit IFN-gamma induced expression of MHC class II (Leroux 2015).

Furthermore, it has been demonstrated that different fungal infectionsare dependent on MHC class II expression. Cryptococos neoformans maycause life threatening brain infections in patients withimmunodeficiencies including HIV. Work in a mouse model of Cryptococosneoformans infection has demonstrated that the activation of microglialcells and their upregulation of MHC class II, in an IFN-gamma dependentmanner, is critical for survival (Zhou 2007).

Therefore, to overcome the immune escaping mechanisms induced by Mtb andother intracellular bacteria, protozoa such as Toxoplasma gondii, orfungi exemplified by Cryptococcus, an increased expression of MHC classII and MHC class I on the cell surface of monocytes, macrophages,microglia, or other infected cells is likely beneficial for immunerecognition and elimination of the pathogen.

Macrolides, such as erythromycin and azithromycin, have been used foryears in the treatment of bacterial infections. Erythromycin is apolyketide natural product macrolide produced by fermentation of theactinomycete Saccharopolyspora erythraea. Azithromycin is asemisynthetic azalide derivative of erythromycin. Many references existdescribing the antibacterial activity of macrolides, such aserythromycin. This antibacterial mechanism is achieved through moleculebinding to the P-site on the bacterial 50S bacterial ribosome, thusinterfering with the tRNA binding.

Many references describe generation of analogues of erythromycin viasemisynthesis and biosynthetic engineering. In particular, methods havebeen described for semisynthetic removal of the glycosyl groups onerythromycin, desosamine and mycarose/cladinose. Further methods havebeen described for biotransformation to add alternative glycosyl groupsto the erythromycin aglycone (e.g. see Gaisser et al., 2000, Schell etal., 2008 and WO2001079520). The main focus of this published work,however, has been to generate antibacterial erythromycin analogues.

DESCRIPTION OF THE INVENTION

Immune stimulating activity from macrolides that lack directantibacterial activity has previously not been reported. Surprisingly,we found that a compound of the invention (compound 1, FIG. 1) had apotent immune stimulating effect on several cell types of the immunesystem. After 24-48 h of in vitro stimulation of peripheral bloodmononuclear cells (PBMC) with 1 μM compound 1 (FIG. 1) the activationmarker CD69 was upregulated on CD4 T cells and B cells (FIG. 2). We alsoobserved upregulation of the MHC class I molecule (HLA-ABC) on T- andB-cells (FIG. 3), indicating an effect on antigen presentation ofantigens derived from intracellular infections. Stimulation of monocytesin the PBMC population with compound 1 led to the upregulation of thecostimulatory molecule CD80 as well as the antigen presenting moleculeMHC class II (HLA-DR) (FIG. 4). Monocytes differentiated intomacrophages also upregulated CD80 in response to stimulation by compound1 (FIG. 5). Furthermore, PBMCs stimulated with compound 1 expressed analtered cytokine profile with increased production of theimmunosuppressive cytokine IL-10, indicating an immune inhibitory effectunder certain conditions (FIG. 6). Further analysis of the immunologicaleffect of compound 1 revealed an altered cytokine driven proliferationprofile of T cells after six days stimulation, measured with flowcytometry (FIG. 7). In addition, virus specific T cell proliferation wasaffected by compound 1. PBMCs from cytomegalovirus (CMV) infected donorscultured in the presence of CMV antigen and compound 1 displayed analtered phenotype of activated CMV specific CD8+ T cells with anincreased expression of IL-7 receptor a (CD127) (FIG. 8). CD127 iscrucial for T cell homeostasis, differentiation and function, andreduced expression correlates with disease severity in HIV and otherchronic viral diseases (Crawley et al Sem Imm 2012). In summary,compound 1 has a surprising ability to specifically activate and modifyan immune response by affecting antigen presentation, co-stimulation andT cell activation and proliferation. In many of these studies, compound2, another related macrolide erythromycin analogue with alteredglycosylation, previously published in Schell et al. 2008 (as compound20), was included as it showed little or no activity in the assays.

Thus, in one aspect of the invention there is provided an immunestimulating macrolide of Formula (I) (also known as compound 1) withoutsubstantial antibacterial activity as defined herein:

Within the scope of the present invention is also compounds of Formula(I) or a pharmaceutically acceptable salt, hydrate, solvate, tautomer,enantiomer or diastereomer thereof.

In another aspect of the invention, there is provided a method forproducing a compound of formula (I), which involves addition of anaglycone with formula II to a culture of a biotransformation strainwhich glycosylates at the 3-hydroxyl position.

In a preferred embodiment of this invention, the biotransformationstrain expresses glycosyltransferases with 70% or more homology toAngMII (SEQ ID no. 1) or AngMIII (SEQ ID no. 2), such as with 75% ormore, with 80% or more, with 90% or more or with 95% or more homologysuch as 100% homology.

The homology between two amino acid sequences or between two nucleicacid sequences is described by the parameter “identity”. Alignments ofsequences and calculation of homology scores may be done using e.g. afull Smith-Waterman alignment, useful for both protein and DNAalignments. The default scoring matrices BLOSUM50 and the identitymatrix are used for protein and DNA alignments respectively. The penaltyfor the first residue in a gap is −12 for proteins and −16 for DNA,while the penalty for additional residues in a gap is −2 for proteinsand −4 for DNA. Alignment may be made with the FASTA package versionv20u6. Multiple alignments of protein sequences may be made using“ClustalW”. Multiple alignments of DNA sequences may be done using theprotein alignment as a template, replacing the amino acids with thecorresponding codon from the DNA sequence. Alternatively, differentsoftware can be used for aligning amino acid sequences and DNAsequences. The alignment of two amino acid sequences is e.g. determinedby using the Needle program from the EMBOSS package (http://emboss.org)version 2.8.0. The substitution matrix used is BLOSUM62, gap openingpenalty is 10, and gap extension penalty is 0.5.

General Chemistry Methods

The skilled person will recognise that the compound of the invention maybe prepared, in known manner, in a variety of ways. The routes below aremerely illustrative of some methods that can be employed for thesynthesis of compounds of formula (I).

In one general route erythromycin A is subjected to semisyntheticmanipulation to generate azithromycin. Methods for this transformationare known (U.S. Pat. Nos. 3,478,014; 4,328,334; and 4,474,768; Glansdorpet al. 2008), though variants on these routes or other routes may beused to the same purpose. The mycarose/cladinose and/or desosamine areremoved by further chemical methods, such as glycoside cleavage.Briefly, in one method the sugars may be removed by treatment with acid.In order to facilitate removal of the amino sugar it is first necessaryto oxidise the dimethylamine to form an N-oxide which is then removed bypyrolysis. The resultant 5-O sugar, and 3-O sugar, can then be removedby acidic degradation. A suitable method is taught by LeMahieu (1974)and Djokic, S., et al., (1988). Finally, the compound is biotransformedusing a bacterial strain which adds the amino sugar.

General use of the Compounds of the Invention

The compound as described herein can be used in medicine, medicalresearch or in the manufacture of a composition for such use.Accordingly, when in the following the term “compound of the invention”is used in connection with medical use or pharmaceutical composition,the term is intended also to include the compound of formula (I)provided that this compound has not been known for such a use.

The compound of the invention is designed in order to minimize directantibacterial effects, but rather focus on immune activating properties.When compound 1 is added to cultures of bacteria E. coli, S. salivarius,L. casei, B. longum or M. luteus no or minimal antibacterial effect isrecognized. The advantage of having a compound with isolated immunestimulatory properties that effect the host cells is that development ofbacterial resistance is avoided. In addition, the well-known side effectof macrolides affecting the gut microbiota, with the risk of overgrowthof Clostridium difficile causing diarrhea and pseudomebraneous colitis,is avoided. Many intracellular pathogens such e.g. Mtb, as well asviruses and cancers have developed mechanisms to avoid immunerecognition, i.e. by down regulating HLA expression, to avoid detectionby T cells. The mechanism of the compound of the intervention rely onthe activation and increased expression of HLA molecules on infectedcells. HLA molecules load and present peptides derived fromintracellular infectious agents in order to present a recognition signalfor T cells allowing elimination of infected cells.

The compound of the invention disclosed herein may be used in thetreatment of intracellular bacterial, fungal, and protozoal infections,such as bacterial infections caused by Mycobacterium tuberculosis,Mycobacteria causing atypical disease, Mycobacterium avium and M.intracellulare (also known as Mycobacterium avium-intracellularecomplex, or MAC), M. kansasii, M. marinum, M. fortuitum, M. gordinae,Mycoplasma pneumoniae, M. genitalium, M. hominis, Ureaplasmaurealyticum, U. parvum, Chlamydophila pneumoniae, and Salmonellatyphimurium, such as protozoal infections caused by Toxoplasma gondii,Plasmodium falciparum, P. vivax, Trypanosoma cruzi, Cryptosporidium, andLeishmania, and such as fungal infections caused by Histoplasmacapsulatum,

Cryptococcus neoformans, and Encephalitozoon cuniculi.

The compound of the invention may be used treatment of intracellularbacterial, fungal, and protozoal infections when these infections occuralone or in association with viral agents or viral disease or inassociation with other causes of primary or secondary immunodeficiency.Causes of primary immunodeficiency include inherited geneticdeficiencies and somatic mutations, whereas secondary immunodeficiencymay be caused by viral infections such as those described above, or byinheritable or non-inheritable conditions such as diabetes mellitus, ormalnutrition, or by agents such as immunodepressants, drug abuse, orother environmental factors.

Moreover, the compound of the invention may be used as a treatment orco-treatment for diseases, disorders, conditions, and symptoms, whereimmune response stimulation is useful, such as in treating patientsinfected with viral agents or with viral diseases such as HIV,Adenovirus, Alphavirus, Arbovirus, Borna Disease, Bunyavirus,Calicivirus, Condyloma Acuminata, Coronavirus, Coxsackievirus,Cytomegalovirus, Dengue fever virus, Contageous Ecthyma, Epstein-Barrvirus, Erythema Infectiosum, Hantavirus, Viral Hemorrhagic Fever, ViralHepatitis, Herpes Simplex Virus, Herpes Zoster virus, InfectiousMononucleosis, Influenza, Lassa Fever virus, Measles, Mumps, MolluscumContagiosum, Paramyxovirus, Phlebotomus fever, Polyoma-virus, RiftValley Fever, Rubella, Slow Disease Virus, Smallpox, Subacute SclerosingPanencephalitis, Tumor Virus Infections, West Nile Virus, Yellow FeverVirus, Rabies Virus and Respiratory Syncitial Virus.

Moreover, the compound of the invention is contemplated to be suitablefor use in the co-treatment of cancer, in particular Adrenal Cancer,Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNSTumors, Breast Cancer, Castleman Disease, Cervical Cancer, Colon/RectumCancer, Endometrial Cancer, Esophagus Cancer, Eye Cancer, GallbladderCancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal StromalTumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, KaposiSarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, AcuteMyeloid Leukemia, Chronic Lymphocytic Leukemia, Acute LymphocyticLeukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia,Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, LungCarcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma,Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer,Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavityand Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, PancreaticCancer, Penile Cancer, Pituitary Tumors, Prostate Cancer,Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Basal andSquamous Cell Skin Cancer, Melanoma, Merkel Cell Skin Cancer, SmallIntestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer,Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer,Waldenstrom Macroglobulinemia, and Wilms Tumor.

Thus, the advantageous properties of the compound of the invention overthe prior art macrolides may include one or more of the following:

-   -   Reduced direct antibacterial activity    -   Improved MHC class I stimulation    -   Improved immunomodulation    -   Improved activation of antigen presenting cells    -   Improved T-cell response    -   Improved antiviral activity    -   Improved MHC class II antigen presentation

Pharmaceutical Compositions Comprising a Compound of the Invention

The present invention also provides a pharmaceutical compositioncomprising the compound of the invention together with one or morepharmaceutically acceptable diluents or carriers. Similarly, the presentinvention also provides a pharmaceutical kit comprising at least onepharmaceutical composition comprising the compound of the inventiontogether with one or more pharmaceutically acceptable excipients. Thepresent invention also relates to cosmetic or veterinary compositionscomprising the compound of the invention together with one or morecosmetically or veterinary acceptable excipients.

The compound of the invention or pharmaceutical, cosmetic, or veterinarycompositions comprising the compound of the invention may beadministered by any conventional route such as but not limited toparenteral, oral, topical, or via a mucosa (including buccal,sublingual, transdermal, vaginal, rectal, nasal, ocular, etc.), via amedical device (e.g. a stent), or by inhalation. The treatment mayconsist of a single administration or a plurality of administrationsover a period of time.

The dosage regimen of the compound of the invention and thepharmaceutical compositions of the invention may be varied depending onthe pharmaceutical properties of the compound or composition inquestion. The dosage regimen may consist of a single administration or aplurality of administrations over one or more periods of time.Administration may be once daily, twice daily, three times daily, fourtimes daily, less frequently, or more frequently, depending on thespecific use, the disease to be treated, and the physical condition andcharacteristics (such as gender, weight, and age) of the patient to betreated. The treatment may also be by continuous administration such ase.g. intravenous administration via a drop or via depots orsustained-release formulations.

Whilst it is possible for the compound of the invention to beadministered as such, it is preferable to present it as a pharmaceuticalcomposition together with one or more pharmaceutically acceptableexcipients. The excipient(s) must be “acceptable” in the sense of beingcompatible with the compound of the invention and not deleterious to therecipients thereof. Examples of suitable excipients are described inmore detail below.

The pharmaceutical compositions may conveniently be presented in asuitable dosage form including a unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Such methodsinclude the step of bringing into association the compound of theinvention with one or more excipients. In general, the pharmaceuticalcompositions are prepared by uniformly and intimately bringing intoassociation the compound of the invention with the excipient(s), andthen, if necessary, shaping the resulting composition into e.g. atablet.

The compound of the invention will normally be administered by anyconventional administration route, such as the oral or any parenteralroute, in the form of a pharmaceutical composition comprising thecompound of the invention, optionally in the form of a pharmaceuticallyacceptable salt, in a pharmaceutically acceptable dosage form. Dependingupon the disorder and patient to be treated, as well as the route ofadministration, the pharmaceutical composition may be administered atvarying doses and/or frequencies.

The pharmaceutical compositions must be stable under the conditions ofmanufacture and storage; thus, if necessary, they should be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. In case of liquid formulations such as solutions, dispersion,emulsions, and suspensions, the excipient(s) can be a solvent ordispersion medium such as but not limited to water, ethanol, polyol(e.g.

glycerol, propylene glycol and liquid polyethylene glycol), vegetableoils, and suitable mixtures thereof, as well as a solvent or dispersionmedium comprising water, ethanol, polyol (e.g. glycerol, propyleneglycol and liquid polyethylene glycol), and vegetable oils.

For example, the compound of the invention may be administered orally,buccally or sublingually in the form of tablets, capsules, films,ovules, elixirs, solutions, emulsions, or suspensions, which may containflavouring or colouring agents.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of thecompound of the invention; as multiple units e.g. in the form of atablet or capsule; as a powder or granules; as a solution or asuspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil liquid emulsion. Thepharmaceutical compositions may also be presented as a bolus, electuary,or paste.

Solutions or suspensions of the compound of the invention suitable fororal administration may also contain one or more solvents includingwater, alcohol, polyol, etc., as well as one or more excipients such aspH-adjusting agents, stabilizing agents, surfactants, solubilizers,dispersing agents, preservatives, flavours, etc. Specific examplesinclude N,N-dimethylacetamide, polysorbate 80, polyethylene glycol, andPhosal 50 PG (which consists of phosphatidylcholine, soya-fatty acids,ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate).The pharmaceutical compositions of the present invention may also be inthe form of emulsions, wherein a compound according to Formula (I) maybe presented in an emulsion such as an oil-in-water emulsion or awater-in-oil emulsion. The oil may be a natural or synthetic oil or anyoil-like substance such as e.g. soy bean oil or safflower oil orcombinations thereof.

Tablets may contain excipients such as microcrystalline cellulose,lactose (e.g. lactose monohydrate or anhydrous lactose), sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine, butylatedhydroxytoluene (E321), crospovidone, hypromellose, disintegrants such asstarch (preferably corn, potato or tapioca starch), sodium starchglycollate, croscarmellose sodium, and certain complex silicates, andgranulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),macrogol 8000, sucrose, gelatin, and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate, andtalc may be included.

A tablet may be made by compression or moulding of a compound of theinvention, optionally with one or more excipients. Compressed tabletsmay be prepared by compressing in a suitable machine the compound of theinvention in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g. povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g. sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose), surface-active agent,and/or dispersing agent. Moulded tablets may be made by moulding in asuitable machine a mixture of the powdered compound of the inventionmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be further treated of processed to provide slowor controlled release of the compound of the invention contained thereinusing, for example, hydroxypropylmethylcellulose in varying proportionsto provide desired release profile.

Solid pharmaceutical compositions of a similar type may also be employedas fillers in gelatin capsules. Preferred excipients in this regardinclude lactose, starch, cellulose, milk sugar, and high molecularweight polyethylene glycols. For aqueous suspensions and/or elixirs, thecompound of the invention may be combined with various sweetening orflavouring agents, colouring matter or dyes, with emulsifying and/orsuspending agents, and with diluents such as water, ethanol, propyleneglycol and glycerin, and combinations thereof.

Pharmaceutical compositions of the invention suitable for topicaladministration in the oral cavity include lozenges comprising thecompound of the invention in a flavoured basis, usually sucrose andacacia or tragacanth; pastilles comprising the compound of the inventionin an inert basis such as gelatin and glycerin, or sucrose and acacia;and mouth-washes comprising the compound of the invention in a suitableliquid carrier.

Pharmaceutical compositions of the invention adapted for topicaladministration may be prepared as ointments, creams, suspensions,lotions, powders, solutions, pastes, gels, impregnated dressings,sprays, aerosols or oils, transdermal devices, dusting powders, and thelike. Such compositions may be prepared via conventional methodscontaining a compound of the invention. Thus, they may also comprisecompatible excipients, such as preservatives, solvents to assist drugpenetration, emollient in creams or ointments, and ethanol or oleylalcohol in lotions. Excipients may constitute from about 1% w/w to about98% w/w of the composition. Preferably, excipients constitute up toabout 80% w/w of the composition. As an illustration only, a cream orointment is prepared by mixing sufficient quantities of hydrophilicmaterial and water, containing from about 5-10% w/w of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions of the invention adapted for transdermaladministration may be presented as discrete patches intended to remainin intimate contact with the epidermis of the recipient for a prolongedperiod of time. For example, the compound of the invention may bedelivered from the patch by iontophoresis.

For applications to external tissues, for example the mouth and skin,the pharmaceutical compositions of the invention are preferably appliedas a topical ointment or cream. When formulated in an ointment, thecompound of the invention may be employed with either a paraffinic or awater-miscible ointment base.

Alternatively, the compound of the invention may be formulated in acream with an oil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms are preparedcomprising the compound of the invention and a sterile vehicle, such asbut not limited to water, alcohols, polyols, glycerine, and vegetableoils, with water being preferred. The compound of the invention,depending on the vehicle and concentration used, can be eithercolloidal, suspended, or dissolved in the vehicle. In preparingsolutions, the compound of the invention can be dissolved in water forinjection and filter sterilised before filling into a suitable vial orampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives, andbuffering agents can be dissolved in the vehicle. To enhance thestability, the pharmaceutical composition can be frozen after fillinginto the vial, and the water may then be removed under vacuum. The drylyophilized powder is then sealed in the vial and an accompanying vialof water for injection may be supplied to reconstitute the liquid priorto use.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, such pharmaceutical compositions can be in the form ofsterile powders for the extemporaneous preparation of such sterileinjectable solutions or dispersions. In all cases, the final injectableform must be sterile and must be effectively fluid for easysyringability.

Parenteral suspensions are prepared in substantially the same manner assolutions, except that the compound of the invention is suspended in thevehicle instead of being dissolved, and sterilization cannot beaccomplished by filtration. The compound of the invention can besterilised by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the pharmaceutical composition to facilitate uniformdistribution of the compound of the invention.

It should be understood that in addition to the ingredients particularlymentioned above, the pharmaceutical compositions of the presentinvention may include other agents conventional in the art having regardto the type of formulation in question. For example, thosepharmaceutical compositions suitable for oral administration may includeflavouring agents. A person skilled in the art will know how to choose asuitable formulation and how to prepare it, e.g. with guidance fromRemington's Pharmaceutical Sciences, 18^(th) edition, Mack PublishingCompany, 1990, or a newer edition. A person skilled in the art will alsoknow how to choose a suitable administration route and dosage.

It will be recognized by a person skilled in the art that the optimalquantity and spacing of individual dosages of the compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects develop,the amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

All % values mentioned herein are % w/w unless the context requiresotherwise.

Definitions

The articles “a”, “an”, and “the” are used herein to refer to one or tomore than one (i.e. at least one) of the grammatical objects of thearticle. By way of example “an analogue” means one analogue or more thanone analogue.

As used herein the term “compound(s) of the invention” are usedinterchangeably and refer to compounds of formula (I).

As used herein the term “direct antibacterial effect” refers to theantibacterial activity of erythromycin and analogues which occursthrough binding to the bacterial rRNA complex. This effect does notrequire presence of any host immune system components and therefore isapparent in standard antibacterial assays such as in vitro MinimumInhibitory Concentration (MIC) assays and disk inhibition assays.

As used herein the term “without substantial antibacterial activity” isintended to mean that the compound of the invention has a MIC valueof >64 μg/ml when tested in accordance with Example 2 herein for itsantibacterial activity in E. coli, S. salivarius, L. casei and B.longum.

The pharmaceutically acceptable salts of the compound of the inventioninclude conventional salts formed from pharmaceutically acceptableinorganic or organic acids or bases as well as quaternary ammonium acidaddition salts. More specific examples of suitable acid salts includehydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric,fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic,tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic,naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic,malic, steroic, tannic and the like. Other acids such as oxalic, whilenot in themselves pharmaceutically acceptable, may be useful in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable salts. Morespecific examples of suitable basic salts include sodium, lithium,potassium, magnesium, aluminium, calcium, zinc,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine and procaine salts.

LEGENDS TO FIGURES

FIG. 1. The structures of the macrolides Erythromycin A, Compound 1,Compound 2, compound 3 and EM703.

FIG. 2. CD69 upregulation on T- and B-cells. PBMC were treated for 24 hwith compound 1, compound 2 and activation controls LPS and IFN-gamma.The expression of the early activation marker CD69 was measured on theCD4+ T cell population (left) and CD19+ B cell population (right) withflow cytometry. Values represents mean fluorescent intensity, MFI, anderror bars standard deviation in the triplicate samples.

FIG. 3. HLA-A,B,C upregulation on T- and B-cells. PBMC were treated for24 h with compounds 1 or 2 and activation controls LPS and IFN-γ. Theexpression of HLA-A,B,C was measured on the CD4+ T cell population(left) and CD19+ B cell population (right) with flow cytometry. Valuesrepresents mean fluorescent intensity, MFI, and error bars standarddeviation in the triplicate samples.

FIG. 4. CD80 and HLA-DR upregulation on blood monocytes. PBMC weretreated for 24 h with compounds 1 or 2 as well as activation controlsLPS and IFN-gamma. The expression of CD80 and HLA-DR was measured on themonocyte cell population with flow cytometry. Values represents meanfluorescent intensity, MFI, and error bars standard deviation in thetriplicate samples.

FIG. 5. CD80 upregulation on blood monocytes. PBMC were treated for 24 hwith compounds 1 or 2 as well as activation control IFN-gamma. Theexpression of CD80 was measured on the monocyte cell population withflow cytometry. Values represents mean fluorescent intensity, MFI, anderror bars standard deviation in the triplicate samples.

FIG. 6. Production of IL-10 from PBMCs after stimulation with compound 1for 48 h or 1 week, measured with ELISA.

FIG. 7. CD4 T cell proliferation after 6 days stimulation with compound1, measured with proliferation dye Celltrace violet (Invitrogen) andflow cytometry. Untreated cells (UNT) or compound 2 were used ascontrols.

FIG. 8. Upregulation of IL-7 receptor a (CD127) on CMV specific CD8 Tcells after incubation with compound 1, measured with flow cytometry.

FIG. 9: Interferon-gamma secretion (as measured by cytometric beadassay) from PBMCs (from a CMV+ donor) grown with CMV peptides in thepresence or absence of compound 1 or 2 for 5 days.

FIG. 10: Interferon-gamma secretion (as measured by cytometric beadassay) from macrophages stimulated with indicated compound for 48 h.

FIG. 11: Chemokine RANTES secretion (as measured by cytometric beadassay) from PBMC or macrophages stimulated with indicated compound for48 h.

FIG. 12: IL12p70 secretion (as measured by cytometric bead assay) fromPBMC or macrophages stimulated with indicated compound for 48 h.

FIG. 13: IL1b secretion (as measured by cytometric bead assay) fromPBMC, macro-phages or CD4 T cells stimulated with indicated compound for48 h.

FIG. 14: % CD25 high cells in blood of C57bl/6 mice injected 24 hpreviously with indicated dose of compound 1. CD25 expression wasmeasured by flow cytometry.

FIG. 15: % MHC class I high CD11b+ cells in spleen of 3 individualC57bl/6 mice injected 24 h previously with indicated compound. MHC classI and CD11b expression was measured by flow cytometry.

EXPERIMENTAL

Materials

Unless otherwise indicated, all reagents used in the examples below areobtained from commercial sources.

Antibodies

Anti-CD80 V450, anti-CD69 PE, anti HLA-DR APC-R700, anti CD127-APC, andanti-Anti-HLA-A,B,C FITC were purchased from BD Biosciences. Celltraceviolet for T cell proliferation assay was purchased from Invitrogen.ELISA antibodies were purchased from BD Biosciences.

Media

RPMI-1640 (Invitrogen) supplemented with 25 mM HEPES, L-glutamine,Sodium pyruvate, 10% fetal bovine serum (Gibco), 100 μg/mL penicillinand 100 μg/mL streptomycin.

General Biology Methods

The effect of the compounds of the invention on immune stimulation maybe tested using one or more of the methods described below:

General Compound Methods

Compound Analysis—Solubility and Stability in Solution

Analysis of Fermentation Broths and Compounds

An aliquot of fermentation broth obtained as described below was shakenvigorously for 30 minutes with an equal volume of ethyl acetate, andthen separated by centrifugation, or the already isolated compounds weredissolved in methanol:water (9:1, 0.1 mg/ml), and then separated bycentrifugation. Supernatants were analysed by LC-MS and LC-MS/MS andchromatography was achieved over base-deactivated Luna C18reversed-phase silica (5 micron particle size) using a Luna HPLC column(250×4.6 mm; Phenomenex (Macclesfield, UK)) heated at 40° C. Agilent1100 HPLC system comprising of quaternary pump, auto sampler, columnoven and diode array detector coupled to a Bruker Esquire ion trap MS.

Mobile phase A=0.1% formic acid in water

Mobile phase B=0.1% formic acid in acetonitrile

Gradient: T=0 min, B=50%; T=4.5 min, B=50%; T=7 min, B=100%; T=10.5 min,B=100%; T=10.75 min, B=50%; T=13 min, B=50%.

Compounds were identified by LC-MS and LC-MS/MS and quantified byLC-MS/MS against an internal standard.

Analysis of Marker Expression by Flow Cytometry

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy donors with Ficoll-Paque density centrifugation. Cells werecultured in complete RPMI-1640 media (Invitrogen) supplemented with 25mM HEPES, L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum,100 μg/mL penicillin and 100 μg/mL streptomycin (Hyclone) for 24-72hours in 37° C., 5% CO₂ and stimulated with and increasingconcentrations of compound 1 and 2. Cells were then washed in PBS andstained with monoclonal antibodies specific for cell surface markers (BDPharmingen) and analysed with flow cytomtery using a BD FACS Canto IIflow cytometer. All samples were tested in duplicates.

Cytomegalovirus (CMV) Cultures

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy CMV positive donors with Ficoll-Paque density centrifugation.The PBMC were labeled with 5 μM celltrace violet (Invitrogen) in PBS for15 minutes and then washed with complete cell culture medium. Thelabeled PBMC was cultured in the presence of a peptide library spanningthe CMV pp65 protein (1 μg peptide/ml, JPT) in AIM-V media (Invitrogen)supplemented with L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovineserum, 100 μg/mL penicillin and 100 μg/mL streptomycin (Hyclone) for 6-8days in 37° C., 5% CO₂. Cell proliferation was assessed with flowcytomtery using a BD FACS Canto II flow cytometer.

ELISA

Supernatant IL-10 was measured with a standard sandwich ELISA (allantibodies from BD Biosciences) after 48 hours and 7 days incubationwith 2.5 μM of compound 1 and 100 U/mL IL-2 (Miltenyi Biotechnologies)in complete RPMI media, 37° C., 5% CO₂

TLR2 Assay

Samples and controls were tested in duplicate on recombinant HEK-293-TLRcell lines using a cell reporter assay at Invivogen using their standardassay conditions. These cell lines functionally over-express human TLR2protein as well as a reporter gene which is a secreted alkalinephosphatase (SEAP). The production of this reporter gene is driven by anNFkB inducible promoter. The TLR reporter cell lines activation resultsare given as optical density values, as compared to negative controls(OD). 20 μl of each test article were used to stimulate the hTLR2reporter cell lines in a 200 μl of final reaction volume. Samples weretested in duplicate, with at least two concentrations tested −20 μM and10 μM.

Assessment of Cell Permeability (Bidirectional)

10 μM Test article was added to the apical (A) surface of Caco-2 cellmonolayers (in HBSS buffer with 0.3% DMSO and 5 μM LY at 37 degrees C.)and compound permeation into the basolateral (B) compartment measuredfollowing 90 minutes incubation. This was also performed in the reversedirection (basolateral to apical) to investigate active transport.LC-MS/MS is used to quantify levels of both the test and standardcontrol compounds. Efflux ratio was calculated by dividing the B to Apermeability by the B to A permeability.Drug permeability: Papp=(VA/(Area×time))×([drug]accepter/(([drug]initial, donor) ×Dilution Factor)

Assessment of Metabolic Stability (Microsome Stability Assay)

Rate of metabolism in microsomes was tested as follows:

Human liver microsomes were diluted with buffer C (0.1 M PotassiumPhosphate buffer, 1.0 mM EDTA, pH 7.4) to a concentration of 2.5 mg/mL.Microsomal stability studies were carried out by adding 30 μL of 1.5 μMcompound spiking solution to wells (1.5 μL of 500 μM spiking solution(10 μL of 10 mM DMSO stock solution into 190 μL ACN to eventuallygenerate final test concentration of 1 uM) and 18.75 μL of 20 mg/mLliver microsomes into 479.75 μL of Buffer C). All samples werepre-incubated for approximately 15 minutes at 37° C. Following this, thereaction was initiated by adding 15 μL of the NADPH solution (6 mM) withgentle mixing. Aliquots (40 μL) were removed at 0, 5, 15, 30 and 45minutes and quenched with ACN containing internal standard (135 μL).Protein was removed by centrifugation (4000 rpm, 15 min) and the sampleplate analysed for compound concentration by LC-MS/MS. Half-lives werethen calculated by standard methods, comparing the concentration ofanalyte with the amount originally present.

EXAMPLES Example 1—Generation of Compound 1

Generation of az-AG

Azithromycin aglycone was generated using methods described in theliterature (Djokic, S., et al., 1988). In brief azithromycin isconverted to azithromycin aglycone by the acidic removal of the 3-O and5-O sugars. The 5-O amino sugar is first oxidised and pyrolyzed tofacilitate cleavage.

Generation of Biotransformation Strains Capable of GlycosylatingErythromycin Aglycones (Erythronolides)

Generation of S. erythraea 18A1 (pAES52)

pAES52, an expression plasmid containing angAI, angAII, angCVI,ang-orf14, angMIII, angB, angMI and angMII along with the actII-ORF4pactI/III expression system (Rowe et al., 1998) was generated asfollows.

The angolamycin sugar biosynthetic genes were amplified from a cosmidlibrary of strain S. eurythermus ATCC23956 obtained from the AmericanType Culture Collection (Manassas, Va., USA). The biosynthetic genecluster sequence was deposited as EU038272, EU220288 and EU232693(Schell et al. 2008).

The biosynthetic gene cassette was assembled in the vector pSG144 asdescribed previously (Schell et al. 2008, ESI), adding sequential genesuntil the 8 required for sugar biosynthesis were obtained, creatingplasmid pAES52.

pAES52 was transformed into strain 18A1 (WO2005054265).

Transformation of pAES52 into S. erythraea 18A1 pAES52 was transformedby protoplast into S. erythraea 18A1 using standard methods (Kieser etal 2000, Gaisser et al. 1997). The resulting strain was designatedISOM-4522, which is deposited at the NCIMB on 24 Jan. 2017 withAccession number: NCIMB 42718.

Generation of S. erythraea SGT2 (pAES54)

pAES54, an expression plasmid containing angAI, angAII, angCVI,ang-orf14, angMIII, angB, angMI and angMII along with the actII-ORF4pactI/III expression system (Rowe et al. 1998) was generated as follows

The angolamycin sugar biosynthetic genes were amplified from a cosmidlibrary of strain S. eurythermus ATCC23956 obtained from the AmericanType Culture Collection (Manassas, Va., USA). The biosynthetic genecluster sequence was deposited as EU038272, EU220288 and EU232693(Schell et al. 2008).

The biosynthetic gene cassette was assembled in the vector pSG144 asdescribed previously (Schell et al. 2008, ESI), adding sequential genesuntil the 8 required for sugar biosynthesis were obtained, creatingplasmid pAES52.

Plasmid pAES54 was made by ligating the 11,541 bp SpeI-NheI fragmentcontaining the actII-ORF4 pactI/III promotor system and the 8 ang geneswas excised from pAES52 with the 5,087 bp XbaI-Spel fragment from pGP9,containing an apramycin resistance gene, oriC, oriT for transfer instreptomycetes and phiBT1 integrase with attP site for integrativetransformation. (The compatible NheI and XbaI sites were eliminatedduring the ligation.)

pAES54 was then transformed into S. erythraea SGT2 (Gaisser et al. 2000,WO2005054265).

Transformation of pAES54 into S. erythraea SGT2

pAES54 was transferred by conjugation into S. erythraea SGT2 usingstandard methods. In brief, E. coli ET12567 pUZ8002 was transformed withpAES54 via standard procedures and spread onto 2TY with Apramycin (50μg/mL), Kanamycin (50 μg/mL), and Chloramphenicol (33 μg/mL) selection.This plate was incubated at 37° C. overnight. Colonies from this wereused to set up fresh liquid 2TY cultures which were incubated at 37° C.until late log phase was reached. Cells were harvested, washed, mixedwith spores of S. erythraea SGT2, spread onto plates of R6 and incubatedat 28° C. After 24 hours, these plates were overlaid with 1 mL ofsterile water containing 3 mg apramycin and 2.5 mg nalidixic acid andincubated at 28° C. for a further 5-7 days. Exconjugants on this platewere transferred to fresh plates of R6 containing apramycin (100 μg/mL).

Alternative biotransformation Strain

Alternatively, BIOT-2945 (Schell et al. 2008) may be used as thebiotransformation strain, as this also adds angolosamine toerythronolides.

Biotransformation of Azithromycin aglycone

Erlenmeyer flasks (250 mL) containing SV2 medium (40 mL) and 8 uLthiostrepton (25 mg/mL) were inoculated with 0.2 mL of spore stock ofstrain ISOM-4522 and incubated at 30° C. and shaken at 300 rpm with a2.5 cm throw for 48 hours.

SV2 Media:

Ingredient Amount glycerol 15 g glucose 15 g soy peptone A3SC 15 g NaCl3 g CaCO₃ 1 g RO water To final volume of 1 L Pre-sterilisation pHadjusted to pH 7.0 with 10M HCl Sterilised by autoclaving @ 121° C., 30minutes

Sterile bunged falcon tubes (50 mL) containing EryPP medium (7 mL) wereprepared and inoculated with culture from seed flask (0.5 mL per falcontube) without antibiotics. The falcons were incubated at 30° C. andshaken at 300 rpm with a 2.5 cm throw for 24 hours.

ERYPP Medium:

Ingredient Amount toasted soy flour (Nutrisoy) 30 g glucose 50 g(NH₄)₂SO₄ 3 g NaCl 5 g CaCO₃ 6 g RO water To final volume of 1 LPre-sterilisation pH adjusted to pH 7.0 with 10M HCl Sterilised in situby autoclaving @ 121° C., 30 minutes Post sterilisation 10 ml/Lpropan-1-ol added

After 24 hours, azithromycin aglycone (0.5 mM in DMSO, 50 uL) was addedto each falcon tube and incubation continued at 300 rpm with a 2.5 cmthrow for a further 6 days.

Isolation of Compound 1

Whole broth was adjusted to pH 9.5 and extracted twice with one volumeof ethyl acetate. The organic layers were collected by aspirationfollowing centrifugation (3,500 rpm, 25 minutes). The organic layerswere combined and reduced in vacuo to reveal a brown gum that containedcompound 1. This extract was partitioned between ethyl acetate (200 ml)and aqueous ammonium chloride (20 ml of a 50% concentrated solution).After separation, the organic layer was extracted with a further volume(200 ml) of the ammonium chloride aqueous solution. The combined aqueouslayers were then adjusted to pH 9.0 with aqueous sodium hydroxide andthen extracted twice with one volume equivalent of ethyl acetate. Theorganic layers were combined and reduced in vacuo to a brown solid. Thisextract was then applied to a silica column and eluted step wise (in 500ml lots) with:

Solvent Hexanes EtOAc MeOH Aq. NH₄OH A 0.499 0.499 0 0.002 B 0.250 0.7480 0.002 C 0 0.998 0 0.002 D 0 0.988 0.01 0.002 E 0 0.978 0.02 0.002 F 00.968 0.03 0.002 G 0 0.958 0.04 0.002

Compound 1 was predominantly in F and G. These solvents were combinedand reduced in vacuo to yield a brown solid containing compound 1. Thismaterial was then purified by preparative HPLC (C18 Gemini NX column,Phenomenex with 20 mM ammonium acetate and acetonitrile as solvent).Fraction containing the target compound were pooled and taken to drynessfollowed by desalting on a C18 SPE cartridge.

Example 2—Assessment of Direct Antibacterial Activity

The bioactivity of macrolide compounds against 4 strains of common gutbacteria (Escherichia coli, Streptococcus salivarius subsp. salivarius,Lactobacillus casei and Bifidobacterium longum subsp. infantis) andcommon mammalian skin isolate Micrococcus luteus, was assessed using theMinimum Inhibitory Concentration (MIC) assay. Bacterial strains werepurchased from DSMZ (Brunswick, Germany) except M. luteus which wasobtained from NCIMB, and stored in 20% glycerol at −80° C. Stocksolutions (100% DMSO) of positive controls (azithromycin anderythromycin), and of test compounds 1 and 2 were diluted in broth toworking stock concentrations of 256 μg/ml (final assay testingconcentration range 128 μg/ml to 0.00391 μg/ml). Stock solutions of allother compounds were diluted in broth to working stock concentrations of128 μg/ml (final assay testing concentration range 64 μg/ml to 0.00195μg/ml). Bacterial strains were cultivated in appropriate broth in ananaerobic chamber at 37° C., except for M. luteus which was incubatedaerobically at 37° C. 18 h cultures were diluted in broth to an OD595 of0.1 and then further diluted 1:10. In 96-well plates, in duplicate, 200μl working stock of test compound was transferred to well 1 and seriallydiluted (1:2) in broth. 100 pl bacterial suspension was aliquoted intoeach well and mixed thoroughly. Appropriate sterility controls wereincluded and plates were incubated in an anaerobic chamber, oraerobically (M. luteus) at 37° C. for 18 h. The MIC was determined to bethe concentration of test compound in the first well with no visiblegrowth.

TABLE 1 Escherichia Streptococcus Lactobacillus BifidobacteriumMicrococcus coli salivarius casei longum luteus Azithromycin  <8 μg/ml<0.5 μg/ml <1.0 μg/ml >64 μg/ml 0.125 μg/ml Erythromycin >64 μg/ml <0.06μg/ml <0.25 μg/ml >64 μg/ml <0.0625 μg/ml Compound 1 >64 μg/ml >64μg/ml >64 μg/ml >64 μg/ml >256 μg/ml EM703 64-128 μg/ml

As can be seen from the data presented in Table 1, compound 1 shows noantibacterial activity against any of the bacterial strains tested,whilst erythromycin and azithromycin show potent activity against anumber of the strains.

Example 3—Assessment of Immune Stimulatory Activity

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy donors with Ficoll-Paque density centrifugation. Cells werecultured in complete RPMI-1640 medium (Invitrogen) supplemented with 25mM HEPES, L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum,100 μg/mL penicillin and 100 μg/mL streptomycin (Hyclone). Cells werestimulated for 24 h (study 1-4) or 48 h to 1 week (study 5) in 37° C.,5% CO₂ with increasing concentrations of compound 1 and 2 in tissueculture plates. The cells were removed from the plate, washed in PBS andanalysed for expression of cell specific surface markers and MHC class Iwith flow cytometry using monoclonal antibodies from BD Pharmingen and aFACS Canto II flow cytometer.

Supernatant IL-10 was measured with a standard sandwich ELISA (allantibodies from BD Biosciences) after 48 hours and 7 days incubationwith 2.5 uM of compound 1 and 100 U/mL IL-2 (Miltenyi Biotechnologies)in complete RPMI media, 37° C., 5% CO₂.

Studyl: After 24 h of in vitro stimulation of peripheral bloodmononuclear cells (PBMC) with 1 μM compound 1 (FIG. 1) the activationmarker CD69 was upregulated on CD4+ T cells and B cells (FIG. 2).

Study 2: We also observed upregulation of the molecule MHC class I(HLA-ABC) on T- and B-cells (FIG. 3), indicating an effect on antigenpresentation of viral antigens.

Study 3: Stimulation of PBMC with compound 1 led to the upregulation ofthe co-stimulatory molecule CD80 as well as the antigen presentingmolecule MHC class II (HLA-DR) on monocytes (FIG. 4).

Study 4: Monocytes differentiated into macrophages also upregulated CD80in response to stimulation by compound 1 (FIG. 5).

Study 5: PBMCs stimulated with compound 1 for 48 h and 7 days expressedan altered cytokine profile with increased production of theimmunosuppressive cytokine IL-10, measured with sandwich ELISA. Thisindicate an immune inhibitory effect under certain conditions (FIG. 6).

Study 6: PBMC were stimulated with compound 1 and cultured in RPMI mediafor 6 days in the presence of IL-2 (Miltenyi Biotechnologies) and CellTrace Violet Dye (Invitrogen). Proliferation was measured with flowcytometry. Analysis of the immunological effect of compound 1 revealedan altered cytokine driven proliferation profile of T cells (FIG. 7).

Study 7: CMV virus specific T cell proliferation was also affected bycompound 1. PBMCs from cytomegalovirus (CMV) infected donors cultured inthe presence of CMV antigen and compound 1 for 6 days displayed analtered phenotype of activated CMV specific CD8+ T cells with anincreased expression of IL-7 receptor a (CD127), measured with flowcytometry (FIG. 8). CD127 is crucial for T cell homeostasis,differentiation and function, and reduced expression correlates withdisease severity in HIV and other chronic viral diseases (Crawley et alSem Imm 2012).

As can be seen, compound 1 has a surprising ability to specificallyactivate and modify an immune response by affecting antigenpresentation, co-stimulation and T cell activation and proliferation. Inmany of these studies, compound 2, another related macrolideerythromycin analogue with altered glycosylation, previously publishedin Schell et al, 2008 (as compound 20), was included and showed littleor no activity in the assays.

Study 8: PBMCs from CMV infected donors cultured in the presence of CMVantigen where either untreated or exposed to compound 1 or compound 2for 3 days. Exposure to compound 1 induced secretion of high levels ofIFN-gamma, whereas antigen culture alone or antigen together withcompounds 2 did not induce IFN-gamma secretion (FIG. 9).

Study 9: Macrophages from healthy donors where exposed to compounds 1 or2 for 48 hours. Only macrophages exposed to compound 1 secretedIFN-gamma whereas untreated macrophages and macrophages exposed tocompound 2 did not secrete IFN-gamma (FIG. 10). Compound 1 is thereforeable to induce IFN-gamma secretion in macrophages from healthy donors.

Study 10: PBMCs and macrophages where exposed to compounds 1 or 2 for 2days (FIG. 11). Basal expression of RANTES in PBMCs was unaffected bycompound 2, whereas compound 1 induced a twofold upregulation ofexpression. Expression of RANTES was miniscule in macrophages, andcompound 1 induced a high expression.

Study 11: PBMCs and macrophages where exposed to compounds 1 and 2 for 2days. PBMCs and macrophages secreted IL-12p70 in response to compound 1,whereas compound 2 failed to induce secretion over untreated cells (FIG.12).

Study 12: PBMCs, macrophages and CD4+ T cells where exposed to compounds1 and 2 for 2 days. IL-1beta secretion was increased by compound 1 inmacrophages and slightly in PBMCs while no IL-1beta was induced in CD4+T cells (FIG. 13).

Study 13: Compound 1 was administered i.v. to C57bl/6 mice at 0.165mg/kg to 5 mg/kg. CD25+ cell abundance was increased in animalsreceiving the highest dose of 5 mg/kg (FIG. 14), as was body weight inthe same group (not shown).

Study 14: Compound 1 or 2 was administered i.v. to C57bl/6 mice. 24 hlater the spleen was removed and MHC class I expression on CD11b+splenocytes was assessed Compound 1 induced an increase in splenocytecells with high MHC I expression, whereas no effect was observed insplenocytes from mice injected with compound 2.

Example 4—Assessment of Activity Against TLR2

Compounds were tested using a TLR2 reporter assay (see general methods)that measured for stimulation of the TLR2 receptor. Stimulatory effectwas measured as an increase in optical density as compared to negativecontrols (OD) due to release of secreted alkaline phosphatase (SEAP) andis shown in table 2.

TABLE 2 OD after OD after OD after addition addition addition of 20 μMof 10 μM of 5 μM test article test article test article Erythromycin A0.045 0.065 0.035 Azithromycin 0.031 0.045 0.029 Compound 2 0.044 0.0100.046 Compound 1 0.458 0.202 0.111 EM703 −0.033 −0.024 −0.040 Compound 3−0.026 −0.015 −0.043

As can be seen, compound 1 stimulated TLR2 at concentrations down to 5μM, whilst erythromycin A, azithromycin and compounds 2 and 3, relatedmacrolide erythromycin analogues with altered glycosylation, previouslypublished in Schell et al, 2008 (as compounds 17 and 20), showed littleor no stimulation at concentrations up to 20 μM.

Example 5—Assessment of caco-2 Permeability

Compounds were tested using a standard caco-2 bidirectional permeabilityassay (see general methods). The data generated is shown in table 3.

TABLE 3 A to B permeability (Papp × 10⁶/cm · s−1) Efflux ratioAzithromycin <0.14 >77.6 Compound 1 0.32 63.4 EM703 <0.15 >108

As can be seen from the data in table 3, Compound 1 is more cellpermeable and has a lower efflux ratio than both Azithromycin and EM703(e.g. see EP1350510).

Example 6—Assessment of Metabolic Stability

The metabolic stability of the compound of the invention was assessed ina standard human microsome stability assay (see general methods).Compounds with longer halflives would be expected to have longerhalf-lives following dosing, which can be useful to allow less frequentdosing. Compounds with shorter half-lives could be useful for use as‘soft drugs’ where the active entity degrades rapidly once entering thepatient's system. The half-life of the compounds assessed in shown intable 4 below:

TABLE 4 T½ (minutes) Azithromycin 245 Erythromycin 31 Compound 1 108EM703 97

REFERENCES

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Gaisser et al. A defined system for hybrid macrolide biosynthesis inSaccharopolyspora erythraea Mol. Micro., 2000; 36(2):391-401

Schell et al. Engineered biosynthesis of hybrid macrolide polyketidescontaining D-an-golosamine and D-mycaminose moieties Org. Biomol. Chem.,2008;6:3315-3327

LeMahieu et al. Glycosidic Cleavage Reactions on Erythromycin A.Preparation of Erythronolide A, J. Med. Chem., 1974, 17(9):953-956

Djokic et al. Erythromycin Series. Part 13. Synthesis and StructureElucidation of 10-Dihydro-10-deoxo-11-methyl-11-azaerythromycin A J.Chem. Res. (5),1988; 5:152-153

Glansdorp et al. Using Chemical Probes to Investigate the Sub-InhibitoryEffects of Azithromycin, Org. Biolmol. Chem., 2008; 208(6): 4120-4124

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All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

The invention claimed is:
 1. A compound, the compound having thestructure of Formula (I):

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the compound according to claim
 1. 3. Thepharmaceutical composition according to claim 2, wherein thepharmaceutical composition comprises one or more pharmaceuticallyacceptable excipients.
 4. A method for treating an intracellularinfection, the method comprising administering to a human or animalsubject in need thereof a therapeutically effective amount of thecompound according to claim
 1. 5. The method according to claim 4,wherein the intracellular infection is selected from intracellularbacterial, intracellular protozoal, and intracellular fungal infections.6. The method according to claim 5, wherein the intracellular infectionis selected from intracellular bacterial infections caused byMycobacterium tuberculosis, Mycobacteria causing atypical disease,Mycobacterium avium and M intracellulare (also known as Mycobacteriumavium-intracellulare complex, or MAC), M kansasii, M marinum, Mfortuitum, M gordinae, Mycoplasma pneumoniae, M genitalium, M hominis,Ureaplasma urealyticum, U. parvum, Chlamydophila pneumoniae, andSalmonella typhimurium.
 7. The method according to claim 5, wherein theintracellular infection is selected from intracellular protozoalinfections caused by Toxoplasma gondii, Plasmodium falciparum, P. vivax,Trypanosoma cruzi, Cryptosporidium, and Leishmania.
 8. The methodaccording to claim 5, wherein the intracellular infection is selectedfrom intracellular fungal infections caused by Histoplasma capsulatum,Cryptococcus neoformans, and Encephalitozoon cuniculi.
 9. A method fortreating or preventing a disease caused by an intracellular infectioncomprising administering to a human or animal subject in need thereof atherapeutically effective amount of the compound according to claim 1.10. A method for preparing the compound as defined in claim 1, themethod comprising adding an aglycone of Formula (II):

to a culture of a biotransformation strain which glycosylates at the3-hydroxyl position of the compound of Formula (II), wherein thebiotransformation strain expresses glycosyltransferases with 70% or morehomology to AngMII (SEQ ID NO: 1) or AngMIII (SEQ ID NO: 2).